Figure 2.

σ^S_db is impaired in DNA promoter binding but not in RNAP core binding. (A) Schematic representation of the four regions of the σ^S protein (17,51) and location of the amino-acid substitutions in σ^S_db in regions involved in binding and melting the -10 promoter element (54,55). (B) The σ^S variants containing one or both substitutions are deficient in DNA promoter binding. When bound to core RNA polymerase (E), σ^S makes sequence-specific promoter contacts and plays a crucial role in DNA melting (54–56). Left panel: electrophoretic mobility shift assay indicating binding of Eσ^S, but not Eσ^S_db, to the katN promoter region. The [³²P]-labeled katN promoter fragment was incubated for 20 min at 37°C with buffer (lane 1), E (lane 2) or holoenzymes reconstituted with His₆-σ^S wild-type (lanes 3 and 7), His₆-σ^S_R141S (lanes 4 and 8), His₆-σ^S_A157T (lanes 5 and 9) and His₆-σ^S_db (lanes 6 and 10) before heparin challenge. The E:σ^S ratios were 1:33 (lanes 3–6) and 1:4 (lanes 7–10). Right panel: KMnO₄ probing of the Eσ^S holoenzymes (E:σ^S of 1:10) on the 5′-[³²P]-labeled template strand katN fragment. KMnO₄ preferentially oxidizes exposed unstacked thymines of RNAP–promoter complexes and gives rise to marked KMnO₄ reactivity at the katN promoter as previously reported (49). The positions of the reactive Ts with respect to the transcription start are indicated (T-11, T-10, T-5, T-4 and T-2). Lane 1: control with no protein; lanes 2 and 6: E His₆-σ^S wild-type; lane 3: E His₆-σ^S_R141S; lane 4: E His₆-σ^S_A157T; lane 5: E His₆-σ^S_db; lane 7: E only. Lane 8 is a G+A reaction performed on the same DNA fragment according to Maxam and Gilbert (58). (C) Distribution of σ^S between free and holoenzyme forms in the wild-type and rpoS_db strains. Whole cell lysates from wild-type strain VF7969 and rpoS_db mutant VF9849 were fractionated by size exclusion chromatography and the relative concentration of RNAP subunits was subsequently analyzed in the fractions by immunoblot using monoclonal antibodies against the β’ and α subunits of RNA polymerase and a polyclonal anti-σ^S antibody. Purified σ^S was used to pinpoint fractions containing free σ^S. The percentage of total σ^S in fractions corresponding to free σ^S was very low (and appeared slightly lower in rpoS_db than in wild-type) suggesting that most σ^S molecules are associated with RNAP in stationary phase. Two independent experiments were performed with similar results (the elution profiles were similar for the wild-type strain and the rpoS_db mutant, and the percentages of total σ^S in fractions 4–8 (bound σ^S) were 69 and 83% for the wild-type strain and 83 and 93% for the rpoS_db mutant, calculated using the IMAGEJ software).